Process for the production of dl glyceraldehyde 3-phosphate and analogs



3,07%,629 PROCESS FOR THE PRGDUCTHGN OF DL GLYQER- ALDEHYDE 3-PHGSPHATE AND ANALOGS Joseph Gold, Berkeley, Caiif. (127 Edgemont Drive, Syracuse 3, NY.) No Drawing. Filed Apr. 3, I958, Ser. No. 727,067 18 Claims. ('Jl. 269-461) My invention relates, in general, to the production of DL glyceraldehyde 3-phosphate and various analogs and, more particularly, to the economical production of such compounds from glycidaldehyde acetal and analogs thereof and inorganic phosphate.

At various stages in the biological processes which relate to the metabolism of carbohydrates and sugars in living cells, e.g., glycolysis, various phosphate intermediates and derivatives including glyceraldehyde 3-phosphate and derivatives thereof are produced or utilized. These materials may therefore be employed in the study of both aerobic and anaerobic biochemical processes as well as in the investigation of the differences which occur in normal and abnormal or pathological cell conditions such as cancer. Identification of the compounds or metabolic derivatives thereof is greatly facilitated by including radioactive phosphorus in the compounds especially when employing analytical techniques such a paper chromatography. In view of the foregoing such materials may be employed as investigative tools for indicating abnormal deposition or metabolic condition. Moreover, the analogs which do not occur in nature and which cannot be metabolized by the cell system can be employed to block or alter the metabolic route so as to inhibit growth of the system. The usual methods of synthesis or production from conventional sources is costly and labeled compounds or the analogs cannot ordinarily be produced from such sources.

It has now been discovered that DL glyceraldehyde 3- phosphate (hereinafter abbreviated GAP) and analogs are produced in a general reaction which occurs between glycidaldehyde acetal and analogs and inorganic orthophosphate salts in aqueous solutions. The GAP or analog is thenceforth economically recovered from the reaction mixture as salts formed with various cations.

Accordingly, it is an object of the invention to provide a process for producing DL glyceraldehyde 3-phosphate and analogs thereof.

Another object of the invention is to provide an economical process for producing DL glyceraldehyde 3-phosphate and analogs thereof by reaction of glycidaldehyde acetal and analogs with inorganic O-phosphate salts including radioactive phosphorus.

Still another object of the invention is to provide a process wherein salts of DL glyceraldehyde 3-phosphate are recovered from the mixture resulting from the reaction of glycidaldehyde acetal and analogs with inorganic O-phosphate salts.

A further object of the invention is to provide synthetic DL glyceraldehyde 3-phosphate analog compositions.

The invention possesses other objects and features of advantage, some of which, with the foregoing, will be set forth in the following description of the preferred form of the invention. It is to be understood, however, that variations in the showing made by the said description may be adopted within the scope of the invention as set forth in the claims.

Glycidaldehyde acetal type compounds of the character described hereinafter and produced by conventional methods are employed as the starting materials in the process of the invention. Such materials are reacted with a salt type reagent, e.g., a phosphate in aqueous solution which adds to the 2,3 epoxy linkage of the starting material yielding a derivative which may be treated or recovered in subsequent operations.

The reaction and reactants which are employed are indicated by the following general equations:

A similar reaction occurs with 2,3 epoxy propanoic acid esters and analogs thereof as indicated in the following equation:

In reactant I, X may be a divalent element or radical such as O or S which is capable of providing an epoxy type linkage between carbon atoms 2 and 3. OR are alkoxy substituents generally of the aliphatic series (but may be of the cyclic variety) containing one or more carbon atoms in the chains, e.g., dimethyl or diethyl acetal, hemiacetal or any other protecting group to provide the glycidaldehyde acetal corresponding to the product desired. Reactant II, ZHPO is generally an acid salt reagent, e.g., a mono-hydrogen O-phosphate salt of alkali or alkaline earth metals as discussed hereinafter, which is capable of breaking the epoxy linkage and adding to the 2,3 carbon atoms.

More specifically, employing glycidaldehyde diethyl acetal as reactant I and disodium hydrogen phosphate as reactant II the reaction may be represented by the equation below. Also shown is an additional equation indicating the reaction between the product DL glyceraldehyde 3-phosphate diethyl acetal and barium acetate which is employed in a product recovery operation discussed more fully hereinafter.

In the foregoing reactions any water soluble monohydrogen O-phosphate salt of alkali and alkaline earth metals may be employed in the place of Na HPO The mono-hydrogen phosphate salts are preferred. Moreover, the phosphorus constituent may include radioactive phosphorus, P whereby the product will be a labeled compound. Also, other water soluble heavy metal salts may be substituted for the barium acetate to combine with the phosphate as well as quaternary ammonium bases such as cyclohexyl amine to form the dicyclohexyl ammonium salt. Other soluble barium salts -including the hydroxide and the chloride may be used likewise. A corresponding series of reactions occur utilizing the epoxy propanoic acid ester as a starting material yielding glyceric acid 3-phosphate acid esters as products.

In practice in carrying out the synthesis process about 0.05 to 0.5 moles of a glycidaldehyde acetal or of the epoxy propanoic acid ester per liter is admixed and dissolved together preferably with a stoichiometric equivalent of the salt such as Na HPO of either natural or radioactive isotopic content in water. If a radioactive iso tope is desired, it is desirable to start with at least a twofold excess of glycidaldehyde acetal to assure more complete utilization of the radioactive phosphate. In the event that the acetal is not completely soluble an oxygenated organic solvent such as dioxane may be substituted for a portion of the water to increase solubility. The aqueous reaction mixture is then refluxed for 48 hours at which time maximum conversion of the reactants occurs. Equivalent results are obtained by reacting for two weeks at room temperature or four days at 37 C. The reaction mixture contains the phosphorylated glycero analog corresponding to the starting material together with unreacted reactants.

Excess salts such as the phosphates may be separated from the reaction mixture by adding a heavy metal cation in the form of a water soluble salt, e.g., BaCl Ba(OI-I) Ba(CH COO) etc., followed by precipitation and filtration therefrom. Subsequent to concentration by evaporation in vacuo to low volume at about 60 C., the heavy metal salt of the DL glyceraldehyde 3-phosphate acetal or the salt of phosphorylated glyceric acid ester also precipitates to a substantial degree from the solution upon cooling. Addition of alcohol-ether mixtures to the solution causes further precipitation of the barium salt of the product. The precipitate is generally filtered from the solution, air dried and washed with alcohol-ether mixtures to remove any residual reaction mixture and excess moisture.

The free phosphorylated acetal or acid ester may ob obtained by dissolving the sodium salt in water and contacting the solution with a cationic exchange resin in the hydrogen form. Cation exchange resins such as Dowex 50 (hydrogen form, 400 mesh) may be employed for this purpose.

The barium salt obtained as indicated in Equation 3 supra serves as a most convenient form for the transportation, storage and distribution of the product. Free GAP acetal or phosphorylated acid ester may as above explained he obtained simply by treating a solution of the salt in water with cation exchange resin in the acid form whereby the barium is replaced by hydrogen from the resin and the barium is absorbed by the resin which is filtered from the solution which contains the phosphoric acid form of the product, i.e., GAP acetal or phosphorylated glyceric acid ester. The free phosphoric acid group of the product is of sufiicient acidic strength so that the acetal is hydrolyzed merely by storage for two days at 37 C. The ester may be converted to any salt by the addition of an equivalent amount of base forming the salt and the alcohol. The solution is then taken down to dryness and the salt of glyceric acid 3-phosphate remains.

Further details of the process of the invention will be apparent from the following specific example:

EXAMPLE Various GAP analogs may be prepared from above indicated glycidaldehyde acetals by substitution into the procedure described hereinafter as applied to glycidaldehyde diethyl acetal.

0.1 mole of glycidaldehyde diethyl acetal was mixed with 27 cc. of H and added slowly to a solution of 0.1 mole of sodium dihydrogen phosphate in 250 cc. of H 0. A small amount of dioxane was added to the reaction mixture as a precaution to insure homogeneity of the system; however, while the precaution was found not to be necessary with the present reagents, with less soluble reagents the dioxane is generally employed. The mixture was then heated at 37 C. and maintained at this temperature for four days wherein the Na HPO reacted at the epoxy sites yielding free DL glyceraldehyde 3-phosphate (disodium salt) diethyl acetal in the solution.

41 g. of Ba (CH COO) in 100 cc. of water was added to the solution to precipitate excess phosphate as Ba (PO The barium phosphate was filtered from the 4, solution, washed with water and filtered to recover occluded product. The filtrates from the washing operations were combined with the reaction mixture filtrate and the combined filtrates were evaporated to a small volume at 60 C. under vacuum applied from an aspirator.

Upon cooling white crystals of the barium salt of DL GAP diethyl acetal separated from the solution and the crystals were filtered from the solution. To assure more complete recovery a mixture of 50% ethanol-50% diethyl ether by volume was added to precipitate additional" barium salt leaving the slight excess of Ba(CH COO) irr the solution and filtrate on repeated filtration. The com-' bined product filter cake was then air dried, washed suc-' cessively with 95% ethanol, absolute ethanol, 75% absolute ethanol25% ether, 25% absolute ethanol-75% ether, dry ether and then dried by evaporation to remove water and residual impurities; however, for many purposes the air dried or less completely washed material is satisfactory. A yield of 6.30 g. or 33.2% of theoretical was obtained.

On an unrecrystallized but washed sample, the product, i.e., the barium salt of DL glyceraldehyde 3-phosphate diethyl acetal, was found to be free of inorganic phosphate, gave a positive test for a protected aldehyde with 2-4 dinitrophenylhydrazine, showed one congruent spot for both organic phosphate and aldehyde on a paper chromatograph, coincided on a paper chromatograph with known glyceraldehyde 3-phosphate and showed a prompt reduction of DPN, diphosphopyridine nucleotide, when tested with the specific crystalline enzyme, glyceraldchyde phosphate dehydrogenase (GAPD-Worthington Biochemi cals Inc).

Analytical results indicated the following:

Carbon, Hydro- Barium, Phosphoperccnt, gen, perpercent ruS, percent cent.

Calculated 22.15 4. 00 36.10 8.18 FOUlll 21. 3. 98 36. 30 8. 00

What is claimed is:

1. In a process for producing a 2, 3 substituted glyceric acid ester type compound, the steps comprising reacting in solution a 2, 3 epoxy propanoic acid ester having a formula wherein X is a divalent linking element selected from the group consisting of O and S and OR is an aliphatic alkoxy protecting group with an inorganic salt ZHPO wherein Z is selected from the group consisting of alkali and alkaline earth metals, to produce a product compound having the formula wherein X is a member selected from the group consisting of O and S and OR is an aliphatic alkoxy protecting group,

'5 with an inorganic salt ZHPO wherein Z is selected from the group consisting of alkali and alkaline earth metals, to produce a product compound having the formula and recovering the product from the reaction mixture.

3. In a process for producing glyceric acid 3-phosphate ester analogs, the step comprising reacting in solution 2, 3 epoxy propanoic acid esters having a formula corresponding to wherein R is an aliphatic alkoxy protecting group with an alkali metal mono-hydrogen O-phosphate salt to produce a glyceric acid ester phosphorylated at the 3-carbon atom.

4. The process as defined in claim 3 wherein said inorganic acid salt includes radio active phosphorous.

5. In a process for producing glyceraldehyde 3-phosphate acetal analogs, the step comprising reacting in solu tion glycidaldehyde acetal having a formula corresponding to wherein R is an aliphatic alkoxy protecting group with an alkali metal mono-hydrogen phosphate salt to produce a glyceraldehyde acetal phosphorylated at the 3-carbon atom.

6. The process as defined in claim 5 wherein said inorganic acid phosphate salt includes radio active phosphorous.

7. In a process for producing glyceric acid 3-phosphate ester analogs, the step comprising reacting in solution 2, 3 epoxy propanoic acid esters having a formula corresponding to wherein R is an aliphatic alkoxy protecting group with an alkali metal mono-hydrogen 0 phosphate salt to produce a glyceraldehyde acetal phosphorylated at the 3-carbon atom.

10. The process as defined in claim 9 wherein said inorganic acid phosphate salt includes radio active phosphorous.

11. In a process for producing 3-phosphate glyceric acid ester analogs, the step comprising reacting in solution 2, 3 epoxy propanoic acid ester with an alkali metal monohydrogen O-phosphate to produce a free 3-phosphate 6 glyceric acid alkyl ester, and recovering said ester from the reaction mixture.

12. In a process for producing glyceraldehyde 3-phosphate analogs, the steps comprising reacting in solution a glycidaldehyde acetal analog with an alkali metal mono hydrogen O-phosphate to produce a free glyceraldehyde 3-phosphate alkyl acetal analog, and recovering said analog from the reaction mixture.

13. In a process for producing 3-phosphate glyceric acid ester analogs, the steps comprising reacting in solution a 2, 3 epoxy propanoic acid aliphatic ester with an alkali metal mono hydrogen O-phosphate to produce a free 3- phosphate glyceric acid alkyl ester analog therein, adding a soluble cation salt selected from the group consisting of Ba and cyclohexyl amine, to the solution to combine with the phosphate of said analog, and recovering the cation derivative of said analog from the reaction mixture.

14. In a process for producing glyceraldehyde 3-phosphate analogs, the steps comprising reacting in solution a glycidaldehyde alkyl acetal analog with an alkali metal mono hydrogen O-phosphate to produce a free glyceraldehyde 3-phosphate alkyl acetal analog therein, adding a soluble cation salt selected from the group consisting of Ba and cyclohexyl amine to the solution to combine with the phosphate of said analog, and recovering the cation derivative of said analog from the reaction mixture.

15. In a process for producing 2, 3 substituted glyceraldehyde acetal and glyceric acid ester type compounds, the steps comprising reacting in solution a reagent selected from the group consisting of O HiO oH(ioR wherein X is a divalent linking element selected from the group consisting of O and S and OR is an alkoxy protecting substituent with a water soluble salt ZHPO where in Z is selected from the group consisting of alkali and alkaline earth metals, in such manner that the epoxy type linkage of X is broken with the -OPO Z portion of the salt adding to the 3 carbon atom and the H atom adding to the X element positioned on the 2 carbon atom in the product and recovering the product from the aqueous reac tion mixture.

16. The barium salt of a 3-phosphate DL glyceric acid ester analog corresponding to the following formula 0 l Ba I. OCHzOH( ]O-R O EICH wherein X is selected from the group consisting of O or S and R is alkyl.

17. A barium salt of a DL glyceraldehyde 3-phosphate acetal analog corresponding to the following formula wherein X is selected from the group consisting of 0 and S and R is alkyl.

18. A barium salt of a DL glyceraldehyde 3-phosphate acetal analog corresponding to the following formula O XH O wherein X is selected from the group consisting of O and S and R is alkyl.

(References on following page) References Cited in the file of this patent Newman: Organic Reactions, John Wiley & Sons,

I D A ATENTS Inc., New York, 1949, page 423. UN ST TES P Ballou et al.: J. Am. Chem. S0c., 76 (3188-3193), 2,466,393 Dickey et a1. Apr. 5, 1949 1954 7 OTHER REFERENCES 5 Ballou et 211.: J. Am. Chem. Soc., 77 (3329333l), 0 1955. 86123211119332 Bull. Soc. Chlrn. France, vol. 31, pa es 848 Loring et all: I. Chem. SUCH P g 3726 Kiessling: Chem. Abst., 29, col. 2510, 1935. 1956- 

15. IN A PROCESS FOR PRODUCING 2, 3 SUBSTITUTED GLYCERALDEHYDE ACETAL AND GLYCERIC ACID ESTER TYPED COMPOUNDS, THE STEPS COMPRISING REACTING IN SOLUTION A REAGENT SELECTED FROM THE GROUP CONSISTING OF
 16. THE BARIUM SALT OF A 3-PHOSPHATE DL GLYCERIC ACID ESTER ANALOG CORRESPONDING TO THE FOLLOWING FORMULA 